Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Engineering Escherichia coli for production of functionalized terpenoids using plant P450s

Abstract

Terpenoids are a highly diverse class of natural products that have historically provided a rich source for discovery of pharmacologically active small molecules1, such as paclitaxel (Taxol) and artemisinin. Unfortunately, these secondary metabolites are typically produced in low abundance in their host organism, and their isolation consequently suffers from low yields and high consumption of natural resources. Furthermore, chemical synthesis of terpenoids can also be difficult to scale for industrial production. For these reasons, an attractive alternative strategy is to engineer metabolic pathways for production of pharmaceuticals or their precursors in a microbial host such as Escherichia coli. A key step is developing methods to carry out cytochrome P450 (P450)-based oxidation chemistry in vivo. Toward this goal, we have assembled two heterologous pathways for the biosynthesis of plant-derived terpenoid natural products, and we present the first examples of in vivo production of functionalized terpenoids in E. coli at high titer using native plant P450s.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: In vivo production of 4 and 6 in E. coli.

Similar content being viewed by others

References

  1. Conolly, J.D. & Hill, R.A. Dictionary of Terpenoids (Chapman & Hall, London, 1991).

    Book  Google Scholar 

  2. Durst, F. & Nelson, D.R. Diversity and evolution of plant P450 and P450-reductases. Drug Metabol. Drug Interact. 12, 189–206 (1995).

    CAS  PubMed  Google Scholar 

  3. Schuler, M.A. & Werck-Reichhart, D. Functional genomics of P450s. Annu. Rev. Plant Biol. 54, 629–667 (2003).

    Article  CAS  Google Scholar 

  4. Chau, M., Jennewein, S., Walker, K. & Croteau, R. Taxol biosynthesis: molecular cloning and characterization of a cytochrome P450 taxoid 7β-hydroxylase. Chem. Biol. 11, 663–672 (2004).

    CAS  PubMed  Google Scholar 

  5. Ortiz de Montellano, P. Cytochrome P450: Structure, Mechanism, and Biochemistry (Springer, New York, 2004).

    Google Scholar 

  6. Sono, M., Roach, M.P., Coulter, E.D. & Dawson, J.H. Heme-containing oxygenases. Chem. Rev. 96, 2841–2887 (1996).

    Article  CAS  Google Scholar 

  7. Wuts, P.G.M. Semisynthesis of taxol. Curr. Opin. Drug Discov. Devel. 1, 329–337 (1998).

    CAS  PubMed  Google Scholar 

  8. Roth, R.J. & Acton, N. A simple conversion of artemisinic acid into artemisinin. J. Nat. Prod. 52, 1183–1185 (1989).

    Article  CAS  Google Scholar 

  9. Wittstock, U. & Halkier, B.A. Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolate. J. Biol. Chem. 275, 14659–14666 (2000).

    Article  CAS  Google Scholar 

  10. Hansen, C.H. et al. Cytochrome P450 CYP79F1 from Arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates. J. Biol. Chem. 276, 24790–24796 (2001).

    Article  CAS  Google Scholar 

  11. Naur, P. et al. CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime. Arch. Biochem. Biophys. 409, 235–241 (2003).

    Article  CAS  Google Scholar 

  12. Haudenschild, C., Schalk, M., Karp, F. & Croteau, R. Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and Saccharomyces cerevisiae. Arch. Biochem. Biophys. 379, 127–136 (2000).

    Article  CAS  Google Scholar 

  13. Bertea, C.M., Schalk, M., Karp, F., Maffei, M. & Croteau, R. Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene. Arch. Biochem. Biophys. 390, 279–286 (2001).

    Article  CAS  Google Scholar 

  14. Schroder, G. et al. Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase. FEBS Lett. 458, 97–102 (1999).

    Article  CAS  Google Scholar 

  15. Irmler, S. et al. Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J. 24, 797–804 (2000).

    Article  CAS  Google Scholar 

  16. Leonard, E., Yan, Y. & Mattheos, M.A.G. Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab. Eng. 8, 172–181 (2006).

    Article  CAS  Google Scholar 

  17. Carter, O.A., Peters, R.J. & Croteau, R. Monoterpene biosynthesis pathway construction in Escherichia coli. Phytochemistry 64, 425–433 (2003).

    Article  CAS  Google Scholar 

  18. Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).

    Article  CAS  Google Scholar 

  19. Newman, J.D. et al. High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol. Bioeng. 95, 684–691 (2006).

    Article  CAS  Google Scholar 

  20. Luo, P., Wang, Y.-H., Wang, G.-D., Essenberg, M. & Chen, X.-Y. Molecular cloning and functional identification of (+)-δ-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1) of cotton sesquiterpene biosynthesis. Plant J. 28, 95–104 (2001).

    Article  CAS  Google Scholar 

  21. Ro, D.K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    Article  CAS  Google Scholar 

  22. Sutter, T.R., Sanglard, D. & Loper, J.C. Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins. J. Biol. Chem. 265, 16428–16436 (1990).

    CAS  PubMed  Google Scholar 

  23. Chen, X.Y., Chen, Y., Heinstein, P. & Davisson, V.J. Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis. Arch. Biochem. Biophys. 324, 255–266 (1995).

    Article  CAS  Google Scholar 

  24. Craft, D.L., Madduri, K.M., Eshoo, M. & Wilson, C.R. Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to α,ω-dicarboxylic acids. Appl. Environ. Microbiol. 69, 5983–5991 (2003).

    Article  CAS  Google Scholar 

  25. Barnes, H.J., Arlotto, M.P. & Waterman, M.R. Expression and enzymatic activity of recombinant cytochrome P450 17-α-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 5597–5601 (1991).

    Article  CAS  Google Scholar 

  26. Schafmeister, C.E., Miercke, L.J.W. & Stroud, R.M. Structure at 2.5 Å of a designed peptide that maintains solubility of membrane proteins. Science 262, 734–738 (1993).

    Article  CAS  Google Scholar 

  27. Sueyoshi, T., Park, L.J., Moore, R., Juvonen, R.O. & Negishi, M. Molecular engineering of microsomal P450 2α-4 to a stable, water-soluble enzyme. Arch. Biochem. Biophys. 322, 265–271 (1995).

    Article  CAS  Google Scholar 

  28. Schoch, G.A., Attias, R., Belghazi, M., Dansette, P.M. & Werck-Reichhart, D. Engineering of a water-soluble plant cytochrome P450, CYP73A1, and NMR-based orientation of natural and alternate substrates in the active site. Plant Physiol. 133, 1198–1208 (2003).

    Article  CAS  Google Scholar 

  29. Roosild, T.P. et al. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307, 1317–1321 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank X.-Y. Chen (Shanghai Institutes for Biological Sciences) for the native CAS and CAH genes, L. Anthony (Amyris Biotechnologies) for the pAM92 plasmid, K. Fisher (Amyris Biotechnologies) for authentic standards, P. Ortiz de Montellano (University of California, San Francisco) for the pCWori plasmid and J. Minshull (DNA 2.0), Y. Yoshikuni, D. Pitera, S. Withers and E. Paradise for helpful discussions. M.C.Y. Chang acknowledges a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund. Funding was provided by the Institute for OneWorld Health through generous support by the Bill and Melinda Gates Foundation.

Author information

Authors and Affiliations

Authors

Contributions

M.C.Y.C. carried out experiments with assistance from R.A.E. and W.T. M.C.Y.C. designed the experiments with input from D.-K.R. M.C.Y.C. and J.D.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jay D Keasling.

Ethics declarations

Competing interests

J.D.K. owns shares of Amyris Biotechnologies, a company that is currently using the technology described here to produce the antimalarial drug artemisinin. Neither Amyris nor the University of California will make any profit (or royalties) from the sale of artemisinin in the developing world.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, M., Eachus, R., Trieu, W. et al. Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nat Chem Biol 3, 274–277 (2007). https://doi.org/10.1038/nchembio875

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio875

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing